The specific -opioid receptor agonist [D-Ala²&#8722;D-Leu⁵]&#8722;enkephalin (DADLE) protects against infarction in the heart when given prior to ischemia. In rabbit this protection leads to phosphorylation of the pro-survival kinases Akt and ERK, and is dependent on transactivation of the epidermal growth factor receptor (EGFR). DADLE reportedly protects rat hearts at reperfusion. We therefore tested whether DADLE at reperfusion could protect isolated rabbit hearts subjected to 30 min of regional ischemia and 120 min of reperfusion, and whether this protection is dependent on Akt, ERK, and EGFR. DADLE (40 nM) was infused for 1h starting 5 min prior to reperfusion and reduced infarct size from 31.0 ± 2.3% in the control group to 14.6 ± 1.6% (p=0.01). This protection was abolished by co-treatment of the metalloproteinase inhibitor (MPI) and the EGFR inhibitor AG1478. In contrast, 20 nM DADLE, although known to be protective prior to ischemia, failed to protect. Western blotting revealed that DADLE's protection was correlated to increase in phosphorylation of the kinases Akt, ERK 1 and 2 in reperfused hearts (2.5 ± 0.5-fold, 1.6 ± 0.2-fold and 2.3 ± 0.7-fold of baseline levels, p<0.05 vs. control). The DADLE-dependent increase in Akt and ERK1/2 phosphorylation were abolished by either MPI or AG1478, confirming a signaling through the EGFR pathway. Additionally, DADLE treatment increased phosphorylation of EGFR (1.4 ± 0.2-fold, p=0.03 vs. control). Thus the -opioid agonist DADLE protects rabbit hearts at reperfusion through activation of the pro-survival kinases Akt and ERK, and dependent on the transactivation of the EGF receptor.